Assessment of Cellular Oxidation using a Subcellular Compartment-Specific Redox-Sensitive Green Fluorescent Protein

Measuring the intracellular oxidation/reduction balance provides an overview of the physiological and/or pathophysiological redox status of an organism. Thiols are especially important for illuminating the redox status of cells via their reduced dithiol and oxidized disulfide ratios. Engineered cysteine-containing fluorescent proteins open a new era for redox-sensitive biosensors. One of them, redox-sensitive green fluorescent protein (roGFP), can easily be introduced into cells with adenoviral transduction, allowing the redox status of subcellular compartments to be evaluated without disrupting cellular processes. Reduced cysteines and oxidized cystines of roGFP have excitation maxima at 488 nm and 405 nm, respectively, with emission at 525 nm. Assessing the ratios of these reduced and oxidized forms allows the convenient calculation of redox balance within the cell. In this method article, immortalized human triple-negative breast cancer cells (MDA-MB-231) were used to assess redox status within the living cell. The protocol steps include MDA-MB-231 cell line transduction with adenovirus to express cytosolic roGFP, treatment with H2O2, and assessment of cysteine and cystine ratio with both flow cytometry and fluorescence microscopy.